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  • br GSLs are tightly associated with

    2020-07-27


    GSLs are tightly associated with membrane proteins and signal transducers in GSL-enriched microdomains (GEMs, also named lipid rafts), and the downstream functional effects of cSrc kinase correlate with Gb3 alterations [46,47]. Silencing the expression of Gb3 synthase with siRNA (siGb3S) to decrease Gb3 synthesis, or disrupting the in-teraction of GSLs (Gb3) with other proteins in GEMs with Shiga toxin 1B subunit (STxB, a specific ligand for Gb3) [48], significantly sup-pressed the levels of pcSrc, β-catenin and METTL3, while increasing pp53 levels, in TP53-Dox Etoposide (Fig. 5A, B). Furthermore, inhibition of cSrc with PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine, a selective Src kinase inhibitor) significantly decreased the levels of cSrc, β-catenin and METTL3 while restoring pp53 levels in TP53-Dox cells. Suppression of the transactivation effects of β-catenin/ Tcf with FH535 ((N-(2-methyl-4-nitro)-2,4-dichlorosulfonamide, a β-
    SW48-Dox cells exposed to Dox
    TP53-Dox cells exposed to Dox _
    METTL3
    PUMA
    Bax
    GAPDH
    A x
    M a
    T p p p
    B
    T
    p
    U
    E
    P
    M
    SW48-Dox exposed to vehicle
    TP53-Dox exposed to vehicle _
    Vehicle PDMPNPC
    Vehicle PDMP NPC
    PUMA
    Bax
    GAPDH
    SW48-Dox cells exposed to vehicle
    Vehicle PDMP NPC
    A x
    M a
    p p p
    B
    p
    U
    P
    (caption on next page)
    Fig. 3. m6A methylation determined missense protein expression in cells carrying p53 R273H mutation. A, Protein expression of p53 and p53 target genes in cells exposed to Dox. Cells were treated with NPC (20 nM), PDMP (5 µM) or vehicle for 5 days, with exposed to Dox (100 nM) during the last 48 h of treatments. Equal amounts of detergent-soluble proteins extracted (50 µg/lane) were resolved using 4–20% gradient SDS-PAGE, and then immunoblotted with corresponding anti-bodies. Top panel, representative Western blots. METTL3, methyltransferase like 3; pp53, phosphorylated p53 (Ser15). Protein levels (bottom panel) are represented as mean ± SD of their optical densities normalized against GAPDH from three settings of blots. *, p < 0.001 compared to SW48-Dox cells; #, p < 0.001 compared to TP53-Dox cells treated with vehicle. B, Protein Expression of p53 and p53 target genes in cells exposed to vehicle. Cells were treated with NPC (20 nM), PDMP (5 µM) or vehicle for 5 days, with exposed to vehicle during the last 48 h of treatments. C, Immunostaining for METTL3 and pp53. Scale bar represents 25 µM in photomicrographs (100x magnification). Red, METTL3-Qdot 605; green, pp53-Alexa Fluor 488. D, Western blotting of p53 and pp53 after immunoprecipitation (IP). Cells were treated with PDMP (5 µM, 6 days) or vehicle, with exposed to Dox (100 nM) during the last 48 h of treatments. Equal amounts of detergent-soluble proteins (500 µg) were precipitated with indicated antibody and then immunoblotted after non-denaturing 12% PAGE resolution. Relative levels of p53 or pp53 are re-presented as means of their optical densities normalized against SW48-Dox cells with vehicle. *, p < 0.001 compared to SW48-Dox with vehicle; **, p < 0.001 compared to TP53-Dox with vehicle. E, MS/MS proteomics analysis of p53. Equal amounts of proteins were precipitated with pp53 antibody, resolved and assessed via mass spectroscopic (MS/MS) analysis.
    catenin/Tcf inhibitor) significantly decreased the protein levels of β-catenin and METTL3, and then increased the levels of pp53, in TP53-Dox cells (Fig. 5A, B). Furthermore, consistent with the significantly increased levels of pp53, TP53-Dox cells were sensitive to Dox after inhibiting ceramide glycosylation with PDMP [19], Gb3 synthesis with siGb3S, GEM interactions with STxB, cSrc kinase activity with PP2, and β-catenin transactivation with FH535, but not upon inhibiting ceramide synthesis with FB1 (Fig. 5C). The IC50 values for Dox in TP53-Dox cells 
    significantly decreased, by more than 12-fold, upon treatment with PDMP, and by approximately 5-fold upon treatments with siGb3S, STxB, PP2 or FH535, as compared to vehicle treatments (Fig. 5C).
    4. Discussion
    Our present study elucidated that methylation of adenosine in TP53 codon R273H RNA transcripts promotes the selection of m6A-R273H
    TP53-Dox Cells
    P
    Vehicle
    Vehicle PDMP NPC
    p53 antibody-IP
    pp53 antibody-IP P
    Western Blot::
    p
    Vehicle
    Vehicle
    Vehicle PDMP
    Vehicle PDMP
    E. MS Assessment of p53 Following IP with pp53 antibody
    Sample
    Protein
    ID probability Peptide sequence
    Mascot Ion score